Steroid hormones are a large family of molecules that play pivotal roles during childhood development. During puberty in humans, elevated secretion of gonadal steroid hormones produces secondary sex characteristics such as breast development and the appearance of facial hair. Because of such important roles of steroid hormones in maturation processes, disruption of steroid hormone signaling during childhood can cause developmental defects that last into adulthood. Understanding the machinery and regulatory mechanisms of steroid hormone signaling in normal as well as pathological conditions, therefore, contributes greatly to the promotion of healthy childhood development. The ultimate goal of this project is to elucidate as-yet-unknown machinery and regulatory mechanisms of steroid hormone release and trafficking, by using the fruitfly Drosophila as a model organism. In order to accomplish this purpose, the Principal Investigator will test the hypothesis that the insect steroid hormone ecdysone is secreted from the steroidogenic tissue in a vesicle-mediated manner, challenging the conventional idea that all steroid hormones are secreted by free diffusion. During the first mentored phase of the project, the Principal Investigator will work closely with his mentor, Michael O'Connor, at the University of Minnesota to develop some key in vitro methods necessary to elucidate his hypothesis. Those methods include the immunohistochemical detection of ecdysone, in vitro transporter assay and in vitro steroidogenic tissue culture. This initial step of the proposed project will help the Principal Investigator master various biochemistry and cell biology techniques required to conduct the next step of the project. During the mentored phase, the Principal Investigator will also undergo extensive training on teaching and scientific communication, which will be helpful in the next independent phase of his career. In the subsequent independent investigator phase, the Principal Investigator will work on the regulatory mechanisms of the putative vesicle-mediated ecdysone release, by screening G protein-coupled receptors working in the steroidogenic tissue. He will also screen for a putative ecdysone importer required for its uptake by peripheral tissues. These approaches should tell us how well this novel machinery of steroid hormone secretion and trafficking is conserved among different organisms. In the long run, the Principal Investigator's work has the potential to shift the paradigm of steroid hormone action and will impact a vast range of research on developmental and disease processes.